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The role of extramembranous cytoplasmic termini in assembly and stability of the tetrameric K(+)-channel KcsA.

Raja M - J. Membr. Biol. (2010)

Bottom Line: The N terminus (1-18 residues) slightly increased tetramer stability via electrostatic interactions in the presence of 30 mol.% acidic phosphatidylglycerol (PG) in phosphatidylcholine lipid bilayer.Furthermore, the N terminus was found to be potentially required for efficient channel (re)assembly.These studies provide new insights into how extramembranous parts play their crucial roles in the assembly and stability of integral membrane protein complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry of Membranes, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands. mraja@pmcol.ualberta.ca

ABSTRACT
Membrane-active alcohol 2,2,2-trifluoroethanol has been proven to be an attractive tool in the investigation of the intrinsic stability of integral membrane protein complexes by taking K(+)-channel KcsA as a suitable and representative ion channel. In the present study, the roles of both cytoplasmic N and C termini in channel assembly and stability of KcsA were determined. The N terminus (1-18 residues) slightly increased tetramer stability via electrostatic interactions in the presence of 30 mol.% acidic phosphatidylglycerol (PG) in phosphatidylcholine lipid bilayer. Furthermore, the N terminus was found to be potentially required for efficient channel (re)assembly. In contrast, truncation of the C terminus (125-160 residues) greatly facilitated channel reversibility from either a partially or a completely unfolded state, and this domain was substantially involved in stabilizing the tetramer in either the presence or absence of PG in lipid bilayer. These studies provide new insights into how extramembranous parts play their crucial roles in the assembly and stability of integral membrane protein complexes.

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Representative Stern–Volmer plots of Trp fluorescence quenching by acrylamide in FL-KcsA (a), ΔN-KcsA (b) and ΔC-KcsA (c) in PC:PG (7:3 mol.%) bilayers. Samples were investigated with (filled circle) or without (filled square) 30 vol.% TFE or upon diluting 30 vol.% TFE-containing samples to achieve a final concentration of 5 vol.% TFE (filled triangle). The slopes of the best fit linear regression lines for each data set (KSV values) are shown in Table 1. d Average fluorescence emission wavelength (λ) as a function of TFE-induced unfolding at indicated TFE concentration followed by refolding upon dilution to reduce TFE concentration to 5 vol.%. Channel refolding upon dilution of 20 vol.% TFE to 5 vol.% resulted in similar emission wavelength (nm) as determined for 0 vol.% TFE for all proteins. Data points correspond to the average ± SD of three or four experiments
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Fig6: Representative Stern–Volmer plots of Trp fluorescence quenching by acrylamide in FL-KcsA (a), ΔN-KcsA (b) and ΔC-KcsA (c) in PC:PG (7:3 mol.%) bilayers. Samples were investigated with (filled circle) or without (filled square) 30 vol.% TFE or upon diluting 30 vol.% TFE-containing samples to achieve a final concentration of 5 vol.% TFE (filled triangle). The slopes of the best fit linear regression lines for each data set (KSV values) are shown in Table 1. d Average fluorescence emission wavelength (λ) as a function of TFE-induced unfolding at indicated TFE concentration followed by refolding upon dilution to reduce TFE concentration to 5 vol.%. Channel refolding upon dilution of 20 vol.% TFE to 5 vol.% resulted in similar emission wavelength (nm) as determined for 0 vol.% TFE for all proteins. Data points correspond to the average ± SD of three or four experiments

Mentions: Within the limits of experimental resolution, changes in fluorescence intensities and red shifts in emission maxima suggested that KcsA might undergo several distinct conformational changes by exposing Trp residues to a more hydrophilic environment during tetramer unfolding. This was assessed in a more direct manner by means of acrylamide, a hydrophilic quencher of Trp fluorescence. This quencher has the advantage that it has a very low permeability to lipid membranes. In addition, no charge interaction takes place between the charged lipid head groups (Lackowicz 1999). The Stern–Volmer quenching plots of FL, ΔN and ΔC-KcsA in the absence and presence of TFE were linear; and representative plots of the effect of 30 vol.% TFE are illustrated in Fig. 6a–c, respectively. The Stern–Volmer constants as a function of TFE concentration are compiled in Table 1.Fig. 6


The role of extramembranous cytoplasmic termini in assembly and stability of the tetrameric K(+)-channel KcsA.

Raja M - J. Membr. Biol. (2010)

Representative Stern–Volmer plots of Trp fluorescence quenching by acrylamide in FL-KcsA (a), ΔN-KcsA (b) and ΔC-KcsA (c) in PC:PG (7:3 mol.%) bilayers. Samples were investigated with (filled circle) or without (filled square) 30 vol.% TFE or upon diluting 30 vol.% TFE-containing samples to achieve a final concentration of 5 vol.% TFE (filled triangle). The slopes of the best fit linear regression lines for each data set (KSV values) are shown in Table 1. d Average fluorescence emission wavelength (λ) as a function of TFE-induced unfolding at indicated TFE concentration followed by refolding upon dilution to reduce TFE concentration to 5 vol.%. Channel refolding upon dilution of 20 vol.% TFE to 5 vol.% resulted in similar emission wavelength (nm) as determined for 0 vol.% TFE for all proteins. Data points correspond to the average ± SD of three or four experiments
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Related In: Results  -  Collection

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Fig6: Representative Stern–Volmer plots of Trp fluorescence quenching by acrylamide in FL-KcsA (a), ΔN-KcsA (b) and ΔC-KcsA (c) in PC:PG (7:3 mol.%) bilayers. Samples were investigated with (filled circle) or without (filled square) 30 vol.% TFE or upon diluting 30 vol.% TFE-containing samples to achieve a final concentration of 5 vol.% TFE (filled triangle). The slopes of the best fit linear regression lines for each data set (KSV values) are shown in Table 1. d Average fluorescence emission wavelength (λ) as a function of TFE-induced unfolding at indicated TFE concentration followed by refolding upon dilution to reduce TFE concentration to 5 vol.%. Channel refolding upon dilution of 20 vol.% TFE to 5 vol.% resulted in similar emission wavelength (nm) as determined for 0 vol.% TFE for all proteins. Data points correspond to the average ± SD of three or four experiments
Mentions: Within the limits of experimental resolution, changes in fluorescence intensities and red shifts in emission maxima suggested that KcsA might undergo several distinct conformational changes by exposing Trp residues to a more hydrophilic environment during tetramer unfolding. This was assessed in a more direct manner by means of acrylamide, a hydrophilic quencher of Trp fluorescence. This quencher has the advantage that it has a very low permeability to lipid membranes. In addition, no charge interaction takes place between the charged lipid head groups (Lackowicz 1999). The Stern–Volmer quenching plots of FL, ΔN and ΔC-KcsA in the absence and presence of TFE were linear; and representative plots of the effect of 30 vol.% TFE are illustrated in Fig. 6a–c, respectively. The Stern–Volmer constants as a function of TFE concentration are compiled in Table 1.Fig. 6

Bottom Line: The N terminus (1-18 residues) slightly increased tetramer stability via electrostatic interactions in the presence of 30 mol.% acidic phosphatidylglycerol (PG) in phosphatidylcholine lipid bilayer.Furthermore, the N terminus was found to be potentially required for efficient channel (re)assembly.These studies provide new insights into how extramembranous parts play their crucial roles in the assembly and stability of integral membrane protein complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry of Membranes, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands. mraja@pmcol.ualberta.ca

ABSTRACT
Membrane-active alcohol 2,2,2-trifluoroethanol has been proven to be an attractive tool in the investigation of the intrinsic stability of integral membrane protein complexes by taking K(+)-channel KcsA as a suitable and representative ion channel. In the present study, the roles of both cytoplasmic N and C termini in channel assembly and stability of KcsA were determined. The N terminus (1-18 residues) slightly increased tetramer stability via electrostatic interactions in the presence of 30 mol.% acidic phosphatidylglycerol (PG) in phosphatidylcholine lipid bilayer. Furthermore, the N terminus was found to be potentially required for efficient channel (re)assembly. In contrast, truncation of the C terminus (125-160 residues) greatly facilitated channel reversibility from either a partially or a completely unfolded state, and this domain was substantially involved in stabilizing the tetramer in either the presence or absence of PG in lipid bilayer. These studies provide new insights into how extramembranous parts play their crucial roles in the assembly and stability of integral membrane protein complexes.

Show MeSH
Related in: MedlinePlus